28S- F2093/3449L 20080617
Date:
Mon, 2008-06-16 23:00
Gene:
Template:
dna
PCR cocktail:
Component | Volume (ul) | Mix (ul) |
Forward primer | 0.5 | |
Reversed primer | 0.5 | |
Buffer | ||
MgCl2 | ||
dNTPs | ||
Taq | ||
DNA (if nested) | 2 | |
ddH2O |
22 | |
Total volume | 25 |
Forward primer(s):
Specimens:
Thermal cycling (abbreviated):
95ºC@4min, 2X (94ºC@30sec,58-54ºC@30sec, 72ºC@60sec),34X (94ºC@30sec,52ºC@30sec, 72ºC@60sec),72ºC@10min
PCR machine:
GeneAmp
Thermal cycling:
Step | Comment | Cycles | Time (sec) | Temperature (°C) |
Initialization | For DNA polymerases that need hot-start heat activation | 1 | ||
Denaturation | DNA melting producing single-stranded DNA | 1 | 240 | 95 |
Amplification | 2 | |||
Denaturation | 30 | 94 | ||
30 | 58-54 | |||
60 | 72 | |||
Amplification | 36 | |||
Denaturation | 30 | 94 | ||
Annealing | 30 | 52 | ||
Extension | 60 | 72 | ||
Final elongation | Any remaining single-stranded DNA is extended | 1 | 600 | 72 |
Hold | 1 | 12 |
Reversed primer(s):